The protein Type III Secretion System (T3SS) is a supramolecular, organic nanomachine that injects bacterial virulence proteins into eukaryotic cells to modulate their physiology for the benefit of the pathogen. It underlies the virulence of numerous medically relevant organisms. A mechanistic understanding of this system is one of the central scientific problems in modern infection biology. At the same time, it is a problem whose solution will also provide important translational benefits, allowing for the development of antivirulence compounds, which target the injection device. Furthermore, the T3SS represents one of the most fascinating examples of a nanomachine. Intellectually, studying the structure, function, and mechanism of the T3SS will impact the understanding of infectious diseases in animals and plants, as well as increase the understanding of nanotechnology and machineries. This grant proposes to solve a specific problem: what is the structural basis for the assembly and function of the T3SS spanning the two membranes of Gram negative bacteria? The project is divided into three specific aims: (1) Optimize and extend current protocols for the expression and purification of T3SS syringe complexes (2) Develop the first recombinant methods for expressing and purifying the secretion complex, and (3) Determine the high-resolution structure of the T3SS nanomachine by X-ray crystallography. To understand the T3SS at the molecular level, high-resolution structural information is required. While moderate resolution electron microscopy (EM) has given an overall view of the system, X-ray crystallographic studies will be required to develop a mechanistic understanding. Such a blueprint for the assembly of the T3SS will be a critical first step towards understanding its function and how to inhibit this function in a therapeutic context.